This invention relates to the regulation of cell differentiation and proliferation, e.g., for treating hyperproliferative skin disorders, such as psoriasis, for enhancing wound healing, and for stimulating hair growth.
Psoriasis is a disease of the epidermis and a major cause of disability and disfigurement for between 1 to 3% of the population of the world. In the United States approximately 2,000,000 to 8,000,000 persons suffer from this disorder, and approximately 100,000 are severely affected.
The disease is diagnosed by the presence of scaling, erythematous lesions on the scalp and extensor aspects of the arms and legs. Psoriatic lesions often are accentuated at sites of repeated trauma such as the elbows and knees. Furthermore, this skin disorder can afflict most of the areas of the skin of some individuals and can also cause internal damage such as arthritis. This disease is characterized by a hyperproliferation of the basal cells (a several fold increase in the number of basal cells of the epidermis). This increase in the basal cell population reduces the turnover time of the epidermis from the normal 27 days to 3-4 days. This shortened interval prevents normal cell maturation and keratinization, and this failure of maturation is reflected in an array of abnormal morphologic and biochemical changes. Numerous cytologic, histologic, histochemical, and biochemical alterations are known to be the result, rather than the cause, of the disease process.
The treatment of psoriasis still remains the province of dermatologists. The most effective treatment for the control of localized psoriasis for most patients is the topical use of corticosteroids with a plastic wrap, ultraviolet light treatments, and sunlight exposure. For certain patients with generalized psoriasis, it has been necessary to use a variety of systemic chemotherapeutic agents, especially methotrexate; the latter has the capacity to inhibit cell replication without a proportionate inhibition of cell function. Photochemotherapy was introduced in 1974, the so-called PUVA treatment. This treatment consists of administering psoralen prior to partial or whole body irradiation with a special light system that emits predominately long wave length ultraviolet light (UV-A).
Recently, it has been appreciated that the skin is not only the site for the synthesis of vitamin D, but is also a target tissue for its biologically active form, 1,25-dihydroxyvitamin D.sub.3 (1,25(OH).sub.2 D.sub.3). A variety of tumor cells (for example, HL-60, U937, and M-1), as well as normal cells (such as activated T-lymphocytes, monocytes, cultured skin fibroblasts, keratinocytes, and cells isolated from the skin of rats, mice, and humans contain a high affinity (1.0.times.10.sup.-10 M), low capacity, protein which is a receptor for 1,25(OH).sub.2 D.sub.3. For tumor cells that possess a receptor for 1,25(OH).sub.2 D.sub.3, the hormone inhibits their proliferation and induces them to differentiate (Suda et al. in Vitamin D: Chemical, Biochemical and Clinical Update (Norman et al., ed.) Walter de Gruyter N. Y. 1985 pp. 187-196). 1,25(OH).sub.2 D.sub.3 inhibits the proliferation of cultured human fibroblasts and keratinocytes and induces keratinocytes to terminally differentiate (Smith et al., J. Invest. Dermatol. 86:709-714, 1986). It has been demonstrated that 1,25(OH).sub.2 D.sub.3 and its analog 1,24-dihydroxyvitamin D.sub.3 are of value in the treatment of psoriasis when provided either orally or topically (Morimoto et al. Brit. J. Dermatol. 115:421-429, 1986; Kato et al. Brit. J. Dermatol. 115, 431-433, 1986).
Aging is associated with a variety of changes in the skin. There is an age-related decrease in epidermal turnover rate of approximately 30 to 50% between the 3rd and 8th decades. Thymidine labeling index (a measure of the proliferative activity of cells) of the epidermis in vivo has been reported to decline nearly 50% with age from approximately 5.1% in 19-25 year old men to approximately 2.85% in 69-85 year old men. Additional investigations have revealed that there is a 100% prolongation in stratum corneum replacement rate in old verses young men (Gilchrest, B. in Skin and Aging Processes, CRC Press, p. 21, 1984).
Parathyroid hormone (PTH), a polypeptide 84 amino acids long, plays an important role in the maintenance of the concentration of ionized calcium in extracellular fluids within normal range. Parathyroid hormone acts directly to raise extracellular calcium by its effects on the bone and kidney, and indirectly by increasing the production of 1,25(OH).sub.2 D.sub.3 to enhance intestinal calcium absorption (Krane S. M. and Potts, J. T. in Harrison's Textbook Principles of Internal Medicine, 9th ed., pp. 1824-1832, 1980). A variety of cells, including kidney cells, lymphocytes, and osteosarcoma cells, possess receptors for parathyroid hormone (Yamamoto, I. et al., J. Clin. Invest. 71:404-407, 1983; Goldring, S. et al., J. Clin. Endo. and Met. 46:425-433, 1978). A variety of in vitro and in vivo tests have been developed to assay for parathyroid hormone. These include the measurement of cyclic AMP production in isolated canine kidney membranes (Nissenson et al., J. Clin. Endo. and Met. 52:840, 1981) and in osteosarcoma cells (Lindall, A. W. et al., J. Clin. Endo. and Met. 57:1007, 1983) and human fibroblasts (Goldring et al., J. Clin. Endo. and Met. 46:425-433, 1978). A cytochemical assay has also been developed to measure glucose-6-phosphate dehydrogenase activity in guinea pig kidney (Goltzman, D. J. et al., Clin. Invest. 65:1309, 1980). In addition, a multiresponse parathyroid hormone assay has been developed to measure both agonist and antagonist properties of parathyroid hormone analogs (Horiuchi, N. et al. Am. J. Physiol. 244:E589-595, 1983).
A variety of PTH analogs have been made. Of interest has been the observation that a 34-residue amino terminal fragment of the PTH molecule [PTH (1-34)] is biologically active in vitro and in vivo (Nussbaum, S. R. et al. J. Prot. Chem. 4:391-406, 1985). Rat PTH (1-34), which has a 5 amino acid sequence difference from the corresponding 1-34 region of both human and bovine PTH, has been found to be 8-10 fold more active than human PTH (1-34) and 2-4 fold more active than bovine PTH (1-34) in the canine adenylate cyclase system (Keutmann, H. T. et al. Endocrinol. 117:1230, 1985). Structure activity studies revealed that PTH-stimulated biologic responses in vitro can be inhibited by [Nle.sup.8, Nle.sup.18, Tyr.sup.34 ] bovine PTH (3-34) PTH amide (bPTH-(3-34)). The parathyroid hormone analog [Tyr.sup.34 ] bovine PTH-(7-34)-amide can inhibit the PTH-mediated elevation of plasma calcium in thyroparathyroidectomized rats in vivo, and is devoid of PTH-like agonist activity (Doppelt, S. H. et al. Proc. Natl. Acad. Sci. USA. 83;7557, 1986).
Recently, it has been found that cultured human keratinocytes make a parathyroid hormone-like protein, referred to herein as "hypercalcemic factor" (Merendino, J. J. et al. Science 231:388-390 1986). Keratinocyte-conditioned medium was harvested from confluent, first passage cultures. Conditioned medium from each of 10 keratinocyte cultures stimulated adenylate cyclase activity in a clonal ROS 17-2.8 cell assay. This biologically active substance is thought to be one of the factors responsible for causing humoral hypercalcemia in patients with a variety of malignancies. This PTH-related protein was isolated from a human lung cancer cell line, and full-length complementary DNA clones encoding it have been inserted into expression vectors used to produce the peptide in mammalian cells. The clones were found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids that has significant homology with parathyroid hormone in the amino terminal region; of the first 16 residues of this protein, 8 of the 16 were found to be identical to human PTH (Suva, L. J. et al. Science 232:893-896, 1987). FIG. 1 herein, reproduced from FIG. 2 of Suva et al., gives the sequences of the two proteins.